1. Field of the Invention
The present invention relates to a novel DNA binding protein which regulates expression of major histocompatibility complex (MHC) class II molecules, DNA sequences which encode the protein, and recombinant expression of the protein.
2. Review of Related Art
Expression of class II major histocompatibility complex (MHC) molecules is usually restricted to cells of the immune system, and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen presenting cell is its ability to express class II MHC molecules. The class II MHC molecules function in the presentation of processed peptides to helper T cells.
The class II region of the human major histocompatibility complex encodes three heterodimeric molecules: HLA-DR, -DQ and -DP, composed of alpha and beta chain polypeptides with an approximate Mr of 60,000. These highly polymorphic molecules determine the ability of an individual to respond to a given antigen, and the molecular basis of this ability lies in the differential capacity of allelic forms of these molecules to bind particular peptides. Peptides derived from extracellular antigens are recognized by helper T cells in the context of these molecules.
Due to the central role these molecules play in the initiation of the immune response, considerable effort is focused on elucidating the mechanisms governing the proper tissue-specific and developmental regulation of the class II MHC genes (Benoist, et al., 1990, Ann. Rev. Immunol., 8:681; Ono, et al., 1991, J. Exp. Med., 173:629). These molecules are expressed constitutively on professional antigen-presenting cells such as macrophages, dendritic cells and B cells, and their biosynthesis is inducible on other cells upon binding of certain lymphokines, such as interferon-gamma, interleukin-4 and tumor necrosis factor alpha, to their respective receptors (Noelle, et al., 1986, J. Immunol., 137:1718; Glimcher, et al., 1992, Ann. Rev. Immunol., 10:13). Class II MHC genes are inactive in plasma cells, and cell fusion experiments indicate that a dominant repressor protein actively inhibits transcription of these genes (Latron, et al., 1988, Proc. Natl. Acad. Sci., USA, 85:2229).
Expression of the class II MHC genes is controlled primarily at the transcriptional level (Ono, et al., 1989, Diabetes, 7:911; Ting, J.P.Y., 1991, Crit. Rev. Immunol., 11:87). Systematic deletion and mutagenesis of the proximal promoters of the human and murine class II genes have identified two highly conserved cis-acting elements called the X and Y boxes that bind several transcription factors that participate in the regulation of these genes (Boss, et al., 1986, Proc. Natl. Acad. Sci., USA, 83:9139; Miwa, et al., 1987, Proc. Natl. Acad. Sci., USA, 84:4939; Viville, et al., 1991, J. Immunol., 146:3211; Klemsz, et al., 1990, Cell, 61:113). These regions are occupied by DNA-binding proteins in class II positive cells but not in class II negative or in certain Bare Lymphocyte Syndrome cell lines (Kara, et al., 1991, Science, 252:709; Wright, et al., 1992, Proc. Natl. Acad. Sci., USA, 89:601).
The X-box is further subdivided into an upstream X1 box 5'CCTAGCAACAGATG3'!(SEQ ID NO:6) and an X2 box 5'CGTCATC3'!(SEQ ID NO:7) located immediately 3' of the X1 box (Latron, et al., 1988). A family of genes encoding X1 box binding proteins have been cloned (RFX1-5) and at least one of these, RFX5, appears to be required for class II MHC gene transcription (Reith, et al., 1988, Cell, 53:897; Reith, et al., 1990, Genes Dev., 4:1528). At least three factors (hXBP1, hXBP2, and c-jun) can interact directly with the X2 box, with the product of the c-fos proto-oncogene being a likely partner (Liou, et al., 1990, Science, 247:1581; Kara, et al., 1990, Mol. Cell. Biol., 10:1347; Anderson, et al., 1990, J. Immunol., 145:3456; Ono, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:4309; Ono, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:4304).
The Y box is in fact an inverted CCAAT box which can bind a multiplicity of factors. Two factors: YB-1 and NF-Y have been implicated in class II MHC gene regulation. YB-1 appears to encode a potent repressor of interferon-gamma induced class II gene expression, while the heterodimeric NF-Y encodes an activator (Didier, et al., 1988, Proc. Natl. Acad. Sci. USA, 85:7322; Zeleznik-Le, et al., 1992, J. Biol. Chem., 267:7677; Li, et al., 1992, J. Biol. Chem., 267:8984). The Y-box may therefore act as a bifunctional cis-element, binding both an activator and repressor of class II MHC gene expression.
Recently, a novel factor (CIITA) required for both constitutive and interferon.gamma. mediated expression of all of the class II MHC genes has been isolated by complementation cloning using a mutant B-lymphoblastoid cell line (Steimle, et al., 1993, Cell, 75:135; Steimle, et al., 1994, Science, 265:106). This factor does not appear to interact directly with the class II MHC proximal promoter, but CIITA transactivation is mediated by the proximal promoter (presumably via protein-protein interactions between CIITA and other class II promoter binding proteins).
A series of classical genetic studies by Accolla and coworkers have previously demonstrated multiple genetic loci that encode either activators or repressors of class II MHC gene expression (reviewed in Glimcher, et al., 1992; Latron, et al., 1988). These studies predicted the existence of two classes of genes termed aIr-1 and sIr-1 that encode either activator(s) or silencer(s) of class II MHC gene expression, respectively. The newly isolated cDNA (CIITA, located on human chromosome 16) appears to encode aIr-1 (Steimle, et al., 1993; Steimle, et al., 1994).
The sIr-1 gene or genes were identified in cell fusion experiments, where factors expressed in the class II negative plasmacytoma cell line P3-U1 were shown to rapidly and dominantly repress class II MHC transcription in the human B cell line Raji. However, neither the sIr-1 gene nor its gene product have been isolated. Since the conserved X1 box of class II MHC genes plays a critical role in the transcriptional regulation of these genes, there is a need for methods of obtaining, in isolated form, the product of the sIr-1 locus.